Hematopoietic stem and progenitor cells are important targets for somatic gene therapy, considering their availability for in vitro manipulation and their enormous biological capacity. The first clinical reports describing successful gene therapeutic correction of severe combined immunodeficiencies by retroviral-vector mediated gene transfer into hematopoietic cells have created new perspectives for the entire field. However, recently at least two cases of secondary leukemias have been reported in which insertional activation of a cellular oncogene by a retrovirally transduced transgene represented the initiating event. We have previously described a similar pathogenesis of leukemic complications in a mouse model of retroviral gene marking. To overcome the current uncertainty which developed in the scientific and regulatory community following these publications, systematic research needs to be conducted to address the frequency of insertional mutagenesis, the impact of vector design, and the role of further contributing factors. Our group has a well-documented expertise in the development of retroviral vectors, selection markers and preclinical assay systems with improved predictive value for hematopoietic gene therapy. On the basis of our findings in preclinical studies and in the literature, we recently developed a classification for side effects related to the genetic manipulation of hematopoietic cells and derived hypotheses addressing the potential for combinatorial interactions of side effects in gene therapy. Three of these hypotheses generate the basis for the present research proposal: (1) In contrast to insertional gene inactivation, insertional gene activation (IGA) may generate more dangerous dominant effects and is expected to be strongly related to the specific architecture and sequence of the inserted transgene. (2) Using retroviral (including lentiviral) delivery technologies, insertional oncogene activation (IOA, a subform of IGA) may be more frequent than previously anticipated. (3) To promote malignant progression of a cell clone with IOA, synergy is required with biological features mediated by transgene expression and systemic conditions of the recipient. The present research proposal addresses these hypotheses by developing novel experimental approaches in cell lines and mouse models of bone marrow transplantation (specific aim 1). Using these models, we will explore the mechanisms of transformation following IOA, by elucidating the impact of vector architecture and its specific regulatory sequences and a potential contributing role of the transgenes involved (specific aim 2). These insights will allow us to develop and evaluate transgene technologies in which the risk of insertional tumorigenesis is significantly reduced, by improving vector design and/or using selectable marker technologies (specific aim 3). We hope that the experimental systems established in this project will have a long-term impact for human gene therapy. Our experiments will also reveal new mechanisms in the genealogy of leukemias, which may have implications for human disease. Quantitative and mechanistic insights into vector safety may become a reality, and the experimental approaches should be applicable for any type of inserting transgene technology developed in the future.